The proposed Program Project aims to understand the replication of prions and the pathogenesis of prion diseases, and to identify therapeutic targets. Prion diseases are probably the most well-understood of the age-dependent neurodegenerative disorders. We propose a series of investigations on the structure of the pathogenic isoform of the prion protein (PrPSc) using cryo-electron crystallography, computational modeling, and small-angle X-ray fiber (Project 1). Studies are also proposed to determine which of the well-established prion strains are suitable for structural analysis by electron crystallography and by fiber diffraction. We propose to use biophysical probes based on solvent accessibility, such as amide-hydrogen exchange or side-chain-thiol exchange, to elucidate the various conformations of the prion protein (PrP) (Project 2) and to study the structural transitions that the PrP can undergo. Misincorporation proton alkyl exchange (MPAX) will allow us to map the solvent-exposed surface of insoluble protein fibrils. Studies on transmembrane forms of PrP in prion replication, as well as their roles in neurodegeneration, are also proposed (Project 3). We hypothesize that a mixture of PrP conformers can be manipulated by previously unappreciated features of PrP's topogenic sequences, and we propose to identify the trans-acting factors with which they interact. In addition, we propose to extend dramatic findings with human (Hu) prions using chimeric Hu/mouse (Mo) PrP transgenes, designated MHu2M (Project 4); Using variants of the MHu2M transgene, we plan to decipher the molecular basis of the species barrier. Recent investigations have defined a limited but feasible study of six Hu residues in the "Hu2" insert of MHu2M that will be altered to determine the effects on incubation times for Hu prions. We will apply this information to the development of rapid transgenic (Tg) mouse models for both Hu prion diseases and chronic wasting disease (CWD) in cervids. We also plan to identify non-PrP host genes that participate in prion propagation/pathogenicity (NPGPPs) (Project 5). The proposed studies employ a unique approach made possible by Random Homozygous Knockout (RHKO), a novel mutational technology that enables the random homozygous inactivation of chromosomal genes in initially susceptible cell populations and the identification and isolation of cells that consequently survive prion-induced lethality. We also propose to knock out both copies of the PrPSc gene in neuroblastoma (N2a) and hypothalamic (GT1) cells that are susceptible to Mo prions and to restore PrPSc formation by expression of mouse PrP or a chimeric PrP as described in Project 4. The identification of NPGPPs for prions, the focus of Project 5, is expected to provide a paradigm for the identification of host genes that affect the pathogenesis of other age-dependent neurodegenerative diseases. The diverse skills, talents, and backgrounds of the investigators involved in the proposed Program offer an unusual opportunity to tackle the molecular mechanisms involved in the conversion of the cellular isoform of the prion protein (PrPc) into PrPSc and to decipher the pathogenesis of prion diseases.